Synthesis of a C-phosphonate mimic of maltose-1-phosphate and inhibition studies on Mycobacterium tuberculosis GlgE

Abstract

The emergence of extensively drug-resistant tuberculosis (XDR-TB) necessitates the need to identify new anti-tuberculosis drug targets as well as to better understand essential biosynthetic pathways. GlgE is a Mycobacterium tuberculosis (Mtb) encoded maltosyltransferase involved in α-glucan biosynthesis. Deletion of GlgE in Mtb results in the accumulation of M1P within cells leading to rapid death of the organism. To inhibit GlgE a maltose-C-phosphonate (MCP) 13 was designed to act as an isosteric non-hydrolysable mimic of M1P. MCP 13, the only known inhibitor of Mtb GlgE, was successfully synthesized using a Wittig olefination as a key step in transforming maltose to the desired product. MCP 13 inhibited Mtb GlgE with an IC50 = 230 ± 24 μM determined using a coupled enzyme assay which measures orthophosphate release. The requirement of M1P for the assay necessitated the development of an expedited synthetic route to M1P from an intermediate used in the MCP 13 synthesis. In conclusion, we designed a substrate analogue of M1P that is the first to exhibit Mtb GlgE inhibition.

Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S

“…Inhibition of this enzyme using 2 α -MTF ( 5 ) and 2-deoxy-2,2-difluoro- β -maltosyl fluoride ( 6 ) was performed by coupling GlgEI-V279 S activity with the commercially available EnzCheck® Phosphate Assay Kit (Life Technologies) 4 . The compounds showed no inhibitory activity below 10 mM concentration.…”

“…4 The assay is performed at room temperature using a Spectra max 340PC microplate reader and a 96 well transparent plate. Each reaction mixture contains 1 mM MESG (20 μL), 0.2 U of PNP (1 μL), 20X reaction buffer (1.0 M Tris-HCl, 20 mM MgCl 2 , PH 7.5, containing 2 mM sodium azide) (5 μL), 50 nM Sco GlgEI-V279S (5 μL), 250 μM sodium salt of α -maltosyl-1-phosphate (3.125 μL), and glycogen (5 μL).…”

“…We have reported the first inhibitors of Mtb GlgE. These include maltose-C-phosphonate (MCP) 4 and 2,5-dideoxy-3- O - α -D-glucopyranosyl-2,5-imino-D-mannitol 5 designed as substrate and transition state inhibitors, respectively. To identify more potent inhibitors we have embarked on a structure-based inhibitor study which utilizes X-ray crystallography to reveal key binding interactions between GlgE and our target ligands.…”

Diversity of phosphorylases in glycoside hydrolase families

“…This is the only phosphorylase that is considered to be involved in the synthesis of glycoside. After the publication of this report, a series of reports considering the inhibitor of GlgE were published with the expectation that they may aid the development of a novel drug for multidrug-resistant strains of M. tuberculosis, the notorious pathogenic bacterium that causes tuberculosis (Kalscheuer et al 2010;Leiba et al 2013;Veleti et al 2014;Syson et al 2014;Sengupta et al 2014Sengupta et al , 2015. The 3D structure of the enzyme from Streptomyces coelicolor is available (Syson et al 2011).…”

α-Glucan biosynthesis and the GlgE pathway in Mycobacterium tuberculosis

“…These include maltose-C-phosphonate and 2,5,dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-Dmannitol, which are substrate analogues and mechanismbased inhibitors respectively [37,38]. A number of potential inhibitors have also been identified by using a docking approach [39][40][41], but these have yet to be verified experimentally.…”